1. Field of the Invention
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid or ribonucleic acid to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research and recombinant DNA technology.
Commonly used methods for detecting specific nucleic acid sequences generally involve immobilization of the target nucleic acid on a solid support. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled nucleotide probe and incubated under hybridizing conditions. After incubation, the solid support is washed to remove any unhybridized probes. The presence of hybridized material on the support is then detected by autoradiography or by spectrometric methods.
The nucleotide probe is typically formed by incorporating a modified nucleotide into an oligonucleotide which facilitates separation or detection of the target sequence. Several factors go into evaluating the suitability of the modified nucleotide as a separation or detection means. The modified nucleotide must contain a unique substituent that is not normally found on nucleotides. The substituent must react specifically with reagents to provide adequate separation or sensitive detection, whether the modified nucleotide is part of a single-stranded or double-stranded polynucleotide. The substituent can not interfere with normal nucleotide interactions so that the modified nucleotide is still able to bind to other bases, i.e., the substituent, of which there may be several on a single modified nucleotide, must not interfere with hybridization. Lastly, the substituent must be bound to the modified nucleotide in such a manner that it will withstand the experimental conditions of hybridization and the subsequent separation and detection.
2. Description of the Related Art
An excellent overview of chemically modified nucleotides is presented in Englisch, et al., Angewandte Chemie (Int. Ed. Engl.) 30(6):613-629 (1991). Chemically labeled nucleotides are also generally disclosed in Stavrianopoulos, et al., U.S. Pat. No. 4,994,373, which discloses labels bound, directly or indirectly by a bridging entity, to a nucleotide probe. Johnston, European Patent Application No. 0,123,300 A2 discloses avidin-biotin-enzyme conjugates used to detect biotinylated nucleotides.
Nucleotide probes can be labeled at the 5' or 3' end such as is disclosed in Brakel, et al. U.S. Pat. No. 5,082,830.
Some methods involve labeling a modified base on one or more nucleotides. Ruth, U.S. Pat. No. 4,948,882 discusses the incorporation of altered purine or pyrimidine bases into chemically synthesized oligonucleotides, where the base has a covalently attached linker arm containing a reporter group. Ward, et al., U.S. Pat. No. 4,711,955 discloses a nucleotide having a detectable moiety chemically bound to a purine, pyrimidine or 7-deazapurine bonded to the 1-position of the sugar moiety. Stavrianopoulos, U.S. Pat. No. 4,707,440 discusses binding biotin or metal chelating compounds to the base of a nucleotide. Heller, et al., U.S. Pat. No. 4,996,143 pertains to the attachment of donor and acceptor fluorophores to a base via linker arms.
Nucleotides can be modified on the sugar moiety, such as is disclosed in Engelhardt, et al., European Patent Application No. 0,302,175 A2, where a substituent is placed on the C2' or the C3' position of the sugar moiety. Various positions on the base and phosphate are also disclosed.
Nucleotides modified at the 1' position are disclosed in Azhayev, et al., Tetrahedron Letters 34(40):6435-6438 (1993) and in Dan, et al., Bioorganic & Medicinal Chemistry Letters 3(4):615-618 (1993).
Altered nucleotides have found utility outside the diagnostics field. For example, nucleosides substituted at the 4' position have been shown to have antiviral activity in Maag, et al., European Patent Application No. 0,457,326 A1, and HIV-inhibitor activity in O-Yang, et al., Tetrahedron Letters 33(1):37-40 (1992) and Maag, et al., Journal Of Medicinal Chemistry 35:1440-1451 (1992). Halazy, et al., European Patent Application No. 0,479,640 A2 discusses nucleosides modified at the phosphate that have antiviral activity. Nucleotides modified at the C5 position of uracil have also shown antiviral and antineoplastic activity in Bergstrom, et al., U.S. Pat. No. 4,247,544.
As described above, most of the modifications fall into one of several categories: modification at the 5' or 3' end; modification of a base; modification of the sugar; and modification at the phosphate. In spite of the advancements in the art, there remains a continuing need to develop improved methods of modifying nucleotides. The end-modification has somewhat limited utility and the base and phosphate modification affect hybridization. Accordingly, there is a continuing need to discover versatile ways to modify nucleotides so that minimal interference with hybridization occurs.